Phosphodiesterases (PDEs) were first detected by Sutherland and co-workers (Rall, et al., J. Biol. Chem., 232:1065-1076 (1958), Butcher, et al., J. Biol. Chem., 237:1244-1250 (1962)). The superfamily of PDEs is subdivided into two major classes, class I and class II (Charbonneau, H., Cyclic Nucleotide Phosphodiesterases: Structure, Regulation and Drug Action, Beavo, J., and Houslay, M. D., eds) 267-296 John Wiley & Sons, Inc., New York (1990)), which have no recognizable sequence similarity. Class I includes all known mammalian PDEs and is comprised of 11 identified families that are products of separate genes (Beavo, et al., Mol. Pharmacol., 46:399-405 (1994); Conti, et al., Endocr. Rev., 16:370-389 (1995); Degerman, et al., J. Biol. Chem., 272:6823-6826 (1997); Houslay, M. D., Adv. Enzyme Regul., 35:303-338 (1995); Bolger, G. B., Cell Signal, 6:851-859 (1994); Thompson, et al, Adv. Second Messenger Phosphoprotein Res., 25:165-184 (1992); Underwood, et al., J. Pharmacol. Exp. Ther., 270:250-259 (1994); Michaeli, et al., J. Biol. Chem., 268:12925-12932 (1993); Soderling, et al., Proc. Natl. Acad. Sci. U.S.A., 95:8991-8996 (1998); Soderling, et al., J. Biol. Chem., 273:15553-15558 (1998); Fisher, et al., J. Biol. Chem., 273:15559-15564 (1998)). Some PDEs are highly specific for hydrolysis of cAMP (PDE4, PDE7, PDE8), some are highly cGMP-specific (PDE5, PDE6, PDE9), and some have mixed specificity (PDE1, PDE2. PDE3, PDE10).
All of the characterized mammalian PDEs are dimeric, but the importance of the dimeric structure for function in each of the PDEs is unknown. Each PDE has a conserved catalytic domain of ˜270 amino acids with a high degree of conservation (25-30%) of amino acid sequence among PDE families, and which is located toward the carboxyl-terminus relative to its regulatory domain. Activators of certain PDEs appear to relieve the influence of autoinhibitory domains located within the enzyme structures (Sonnenberg, et al., J. Biol. Chem., 270:30989-31000 (1995); Jin, et al., J. Biol. Chem., 267:18929-18939 (1992)).
PDEs cleave the cyclic 2′-3′ nucleotide phosphodiester bond between the phosphorus and oxygen atoms at the 3′-position with inversion of configuration at the phosphorus atom (Goldberg, et al., J. Biol. Chem., 255:10344-10347 (1980); Burgers, et al., J. Biol. Chem., 254:9959-9961 (1979)). This apparently results from an in-line nucleophilic attack by the OH of ionized H2O. It has been proposed that metals bound in the conserved metal binding motifs within PDEs facilitate the production of the attacking OH− (Francis, et al., J. Biol. Chem., 269:22477-22480 (1994)). The kinetic properties of catalysis are consistent with a random order mechanism with respect to cyclic nucleotide and the divalent cations(s) that are required for catalysis (Srivastava, et al., Biochem. J., 308:653-658 (1995)). The catalytic domains of all known mammalian PDEs contain two sequences (HX3HXn(E/D)) arranged in tandem, each of which resembles the single Zn2+-binding site of metalloendoproteases such as thermolysin (Francis, et al., J. Biol. Chem., 269:22477-22480 (1994)). PDE5 specifically binds Zn2+, and the catalytic activities of PDE4, PDE5, and PDE6 are supported by submicromolar concentrations of Zn2+ (Francis, et al., J. Biol. Chem., 269:22477-22480 (1994); Percival, et al., Biochem. Biophys. Res. Commun., 241:175-180 (1997)). Whether each of the Zn2+-binding motifs binds Zn2+ independently or whether the two motifs interact to form a novel Zn2+-binding site is not known. The catalytic mechanism for cleaving phosphodiester bonds of cyclic nucleotides by PDEs may be similar to that of certain proteases for cleaving the amide ester of peptides, but the presence of two Zn2+ motifs arranged in tandem in PDEs is unprecedented.
The group of Sutherland and Rall (Berthet, et al., J. Biol. Cem., 229:351-361 (1957)), in the late 1950s, was the first to realize that at least part of the mechanism(s) whereby caffeine enhanced the effect of glucagon, a stimulator of adenylyl cyclase, on cAMP accumulation and glycogenolysis in liver involved inhibition of cAMP PDE activity. Since that time chemists have synthesized thousands of PDE inhibitors, including the widely used 3-isobutyl-1-methylxanthine (IBMX). Many of these compounds, as well as caffeine, are non-selective and inhibit many of the PDE families. One important advance in PDE research has been the discovery/design of family-specific inhibitors such as the PDE4 inhibitor, rolipram, and the PDE5 inhibitor, sildenafil.
Precise modulation of PDE function in cells is critical for maintaining cyclic nucleotide levels within a narrow rate-limiting range of concentrations. Increases in cGMP of 2-4-fold above the basal level will usually produce a maximum physiological response. There are three general schemes by which PDEs are regulated: (a) regulation by substrate availability, such as by stimulation of PDE activity by mass action after elevation of cyclic nucleotide levels or by alteration in the rate of hydrolysis of one cyclic nucleotide because of competition by another, which can occur with any of the dual specificity PDEs (e.g. PDE1, PDE2, PDE3); (b) regulation by extracellular signals that alter intracellular signaling (e.g. phosphorylation events, Ca2+, phosphatidic acid, inositol phosphates, protein-protein interactions, etc.) resulting, for example, in stimulation of PDE3 activity by insulin (Degerman, et al., J. Biol. Chem., 272:6823-6826 (1997)), stimulation of PDE6 activity by photons through the transducin system (Yamazaki, et al., J. Biol. Chem., 255:11619-11624 (1980)), which alters PDE6 interaction with this enzyme, or stimulation of PDE1 activity by increased interaction with Ca2+/calmodulin; (c) feedback regulation, such as by phosphorylation of PDE1, PDE3, or PDE4 catalyzed by PKA after cAmP elevation (Conti, et al., Endocr. Rev., 16:370-389 (1995); Degerman, et al., J. Biol. Chem., 272:6823-6826 (1997); Gettys, et al., J. Biol. Chem. 262:333-339 (1987); Florio, et al, Biochemistry, 33:8948-8954 (1994)), by allosteric cGMP binding to PDE2 to promote breakdown of cAMP or cGMP after cGMP elevation, or by modulation of PDE protein levels, such as the desensitization that occurs by increased concentrations of PDE3 or PDE4 following chronic exposure of cells to cAMP-elevating agents (Conti, et al., Endocr. Rev., 16:370-389 (1995), Sheth, et al., Throm. Haemostasis, 77:155-162 (1997)) or by developmentally related changes in PDE5 content. Other factors that could influence any of the three schemes outlined above are cellular compartmentalization of PDEs (Houslay, M. D., Adv. Enzyme Regul., 35:303-338 (1995)) effected by covalent modifications such as prenylation or by specific targeting sequences in the PDE primary structure and perhaps translocation of PDEs between compartments within a cell.
Within the PDE superfamily, four PDEs (PDE2, PDE5, PDE6, and PDE10) of the 10 families contain highly cGMP-specific allosteric (non-catalytic) cGMP-binding sites in addition to a catalytic site of varying substrate specificity. Each of the monomers of these dimeric cGMP-binding PDEs contains two homologous cGMP-binding sites of ˜110 amino acids arranged in tandem and located in the amino-terminal portion of the protein (Charbonneau, H., Cyclic Nucleotide Phosphodiesterases: Structure, Regulation and Drug Action, Beavo, J., and Houslay, M. D., eds) 267-296 (1990); McAllister-Lucas, et al., J. Biol. Chem., 270:30671-30679 (1995)). In PDE2, binding of the cGMP to these sites stimulates the hydrolysis of cAMP at the catalytic site (Beavo, et al., Mol. Pharmacol., 46:399-405 (1994)). PDE2 hydrolyzed cGMP as well as cAMP, and cGMP hydrolysis is stimulated by cGMP binding at the allosteric sites in accordance with positively cooperative kinetics (Manganiello, et al., Cyclic Nucleotide Phosphodiesterases: Structure, Regulation, and Drug Action, Beavo, J., and Houslay, M. D., eds, 61-85 John Wiley & Sons, Inc., New York (1990)). This could represent a negative feedback process for regulation of tissue cGMP levels (Manganiello, et al., Cyclic Nucleotide Phosphodiesterases: Structure, Regulation, and Drug Action, Beavo, J., and Houslay, M. D., eds, 61-85 John Wiley & Sons, Inc., New York (1990)), which occurs in addition to the cross-talk between cyclic nucleotide pathways represented by cGMP stimulation of cAMP breakdown. Binding of cGMP to the allosteric sites of PDE6 has not been shown to affect catalysis, but this binding may modulate the interaction of PDE6 with the regulatory protein, transducin, and the inhibitory y subunit of PDE6 (Yamazaki, et al., Adv. Cyclic Nucleotide Protein phosphorylation Res., 16:381-392 (1984)).
The PDE4 subfamily is comprised of 4 members: PDE4A (SEQ ID NO:14), PDE4B (SEQ ID NO:12), PDE4C (SEQ ID NO: 15), and PDE4D (SEQ ID NO:13) (Conti et al. (2003) J Biol Chem. 278:5493-5496). The PDE4 enzymes display a preference for cAMP over cGMP as a substrate. These enzymes possess N-terminal regulatory domains that presumably mediate dimerization, which results in optimally regulated PDE activity. In addition, activity is regulated via cAMP-dependent protein kinase phosphorylation sites in this upstream regulatory domain. These enzymes are also rather ubiquitously expressed, but importantly in lymphocytes.
Inhibitors of the PDE4 enzymes have proposed utility in the treatment of inflammatory diseases. Knockout of PDE4B results in viable mice (Jin and Conti (2002) Proc Natl Acad Sci USA, 99, 7628-7633), while knockout of PDE4D results in reduced viability (Jin et al. (1999) Proc Natl Acad Sci USA, 96, 11998-12003). The PDE4D knockout genotype can be rescued by breeding onto other background mouse strains. Airway epithelial cells from these PDE4D knockout embryos display greatly reduced hypersensitivity to adrenergic agonists, suggesting PDE4D as a therapeutic target in airway inflammatory diseases (Hansen et al. (2000) Proc Natl Acad Sci USA, 97, 6751-6756). PDE4B-knockout mice have few symptoms and normal airway hypersensitivity.
By contrast, monocytes from the PDE4B knockout mice exhibit a reduced response to LPS (Jin and Conti (2002) Proc Natl Acad Sci USA, 99, 7628-7633). This suggests that a PDE4B compound with selectivity versus PDE4D could exhibit anti-inflammatory activity with reduced side-effects.
Crystal structures of PDE4B (Xu et al. (2000) Science, 288, 1822-1825) and PDE4D (Lee et al. (2002) FEBS Lett, 530, 53-58) have been reported in the literature. The PDE4B structure was solved without ligand present in the active site, so information about active site properties was limited to determination of two metal ion sites (presumably zinc and magnesium). A binding mode for cAMP was proposed based on computational modeling. Accordingly, there is need in the art for more potent and specific inhibitors and modulators of PDE4B and methods for designing them.